The long-term objective of this project is to elucidate the cellular mechanisms of learning and memory. The withdrawal reflex of the marine snail Aplysia californica constitutes a useful model system for this purpose. First, the reflex exhibits several forms of learning, including short-term habituation, short- and long-term dishabituation/sensitization, and classical conditioning. Second, the neuronal circuit which mediates the withdrawal reflex is relatively well-understood, particularly the monosynaptic component of the circuit. Several short- and long-term plastic phenomena, which parallel, and may have mechanistic roles in behavioral modification of withdrawal, have been demonstrated at the sensorimotor synapse involved in withdrawal. Thus, the sensorimotor connection is a likely locus for learning-related cellular changes. Finally, the monosynaptic component of the withdrawal reflex can be reconstituted in dissociated cell culture. Such in vitro synapses greatly facilitate experimental dissection of the various cellular modifications, presynaptic and postsynaptic, which contribute to learning. These in vitro synapses are ideal, moreover, for studies involving optical recording and video microscopy. The proposed experiments utilize Aplysia sensorimotor synapses in dissociated cell culture for examining changes in morphology and in intracellular calcium, associated with plastic changes at this synapse. Calcium, because of its properties as a carrier of positive charge within neuronal membranes, as a trigger for exocytotic transmitter release, and as an activator of a variety of cytoplasmic and nuclear responses in neurons, is thought to play a significant role in synaptic plasticity. Optical recording techniques, together with video fluorescence microscopy, will be utilized to determine the specific changes in structure and in intracellular calcium that take place during a variety of forms of plasticity at in vitro sensorimotor synapses. In particular, changes in the dendritic structure of the motor neuron during long-term facilitation will be determined. How levels of calcium are altered in presynaptic varicosities or postsynaptic dendrites during homosynaptic depression, posttetanic potentiation, presynaptic facilitation, and activity-dependent facilitation will also be examined. Finally, the regulation of presynaptic transmitter release by the postsynaptic cell, as well as the cellular nature of this regulation and its implications for plasticity will also be studied. It is expected that the proposed research will significantly contribute to an understanding of the cellular substrates of learning and memory in nervous systems generally, and that the work may ultimately provide insights into memory-associated disease, such as Alzheimer's.